1. Field of the Invention
This invention relates generally to a method of inducing embryonic stem cells. Particularly, the present invention relates to a method for inducing embryonic stem cells into pancreatic cells, and a kit with the same use.
2. Description of the Prior Art
As the most common metabolic disorder diseases in human, Diabetes Mellitus affects 4 to 5% of the world population. The number of patients with diabetes is predicted to exceed 350 million by 2010. Type I diabetes mellitus results from the autoimmune destruction of the β cells in pancreatic islets. Many research groups are therefore exploring ways to replace these destroyed insulin-producing cells. Until now, pancreatic islet cell transplantation is the only effective approach to cure type I diabetes in addition to insulin injection(Serup P, Madsen O D, Mandrup-Poulsen T. Islet and stem cell transplantation for treating diabetes. BMJ 2001; 322: 29-32). However, this method could not be widely utilized due to the severe shortage of transplantable donor islets.
Functional β cell obtained from embryonic stem (ES) cells has been considered to solve the problem of the shortage of transplantable islets: ES cells have been shown to be able to differentiate into pancreatic islet-like clusters, especially pancreatic β cells' (Soria B, Roche E, Berna G, et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000; 49(2):157-162; Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001; 292:1389-1394; Assady S, Maor G, Amit M, et al. Insulin production by human embryonic stem cells. Diabetes 2001; 50(8):1691-169.). Soria et al., for the first time, successfully induced ES cells to differentiate into pancreatic β cells by a cell-trapping system (Soria B, Roche E, Berna G, et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000; 49(2):157-162). This method, however, is a complicated process involving genetic manipulation. Lumelsky et al (Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001; 292:1389-1394) came up with a five-stage method to induce ES cells to differentiate into insulin-producing islet-like structures without genetic modification. Hori et al (Hori Y, Rulifson I C, Tsai B C, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci USA 2002; 99(25):16105-16110) and Blyszczuk et al (Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc NatI Acad Sci USA 2003; 100(3):998-1003) improved Lumelsky's five-stage method by adding growth inhibitor LY294002 or overexpressing pax4 gene. Nonetheless, all these induction approaches are complicated in certain aspects and take a long period of time. Hansson et al, (Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci USA 2003; 100(3):998-1003), after using the five-stage method, found that the absorbance of insulin from the culture medium by ES cell may lead to false positive result. Therefore, what is needed is to find the specific induction factors that can induce ES cells to differentiate into pancreatic β cells in an easy and fast approach. Some factors have been reported to have the ability to promote the differentiation of definitive endoderm. Activin A, a member of TGF-β superfamily, is critical for mesoderm and endoderm formation during gastrulation. When used at a high concentration, it substantially induces endoderm formation (Kumar M, Jordan N, Melton DA, et al. Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Developmental Biology 2003; 259: 109-122; Hill CS. TGF-β signalling pathways in early Xenopus development. Curr Opin Genet Dev 2001; 11(5):533-540). All-trans retinoic acid (RA) is a well-characterized signaling molecule that acts in anteroposterior patterning of neuroectoderm and mesoderm in vertebrates (Maden M. Role and distribution of retinoic acid during CNS development. Int Rev Cytol 2001; 209:1-77). Current evidence indicates that RA is also involved in the regulation of the embryonic endoderm differentiation pattern especially in the early pancreas bud formation and it can also improve insulin expression in pancreatic β cells and INS-1 cell line (Stafford D, Prince V E. Retinoic Acid signaling is required for a critical early step in Zebrafish pancreatic development. Current Biology 2002; 12(14):1215-1220; Blumentrath J, Neye H, Verspohl E J. Effects of retinoids and thiazolidinediones on proliferation, insulin release, insulin mRNA, GLUT2 transporter protein and mRNA of INS-1 cells. Cell Biochem Funct 2001; 19:159-169). It has been demonstrated that the combination of activin A and RA was able to induce Xenopus presumptive ectoderm region of the blastula to differentiate into pancreatic insulin-positive cells (Moriya N, Komazaki S, Takahashi S, et al. In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid. Develop Growth Differ 2000; 42:593-602).